The hepatitis C virus (HCV) specifically targets the human protein synthesis machinery to translate its genomic RNA via an internal initiation mechanism. To position the start codon for initiation, the HCV genome encodes an Internal Ribosome Entry Site (IRES) RNA element that binds to the 40S ribosomal subunit and to translation initiation factor eIF3. However, the specific molecular interactions that control the initiation steps from the IRES remain poorly defined. Furthermore, the mechanisms by which microRNA binding enhances viral RNA stability and replication are also unclear. We propose to use biochemical and x-ray crystallographic methods to identify and define the specific interactions between the HCV IRES, eIF3 and the 40S ribosomal subunit that are important for HCV IRES-dependent translation initiation. We will also determine the structural basis and functional implications of miR-122 binding to the 5'-end of the HCV genomic RNA. The specific aims of the proposal are: I. Determine molecular structures of the HCV IRES RNA alone and in complex with the human ribosome. Structures of the HCV IRES pseudoknot domain and of the IRES bound to the human 40S subunit will provide unprecedented insights into how the IRES controls translation initiation. II. Test the mechanism of microRNA-induced HCV RNA folding and factor recruitment. The biochemistry and structural biology of miR-122 bound to the 5'-terminus of the HCV genomic RNA will help to explain the importance of this interaction for HCV viability in human cells. III. Determine the structural basis for HCV IRES interactions with eIF3. Recombinant human eIF3 expressed in E. coli will be used to find the minimal components of eIF3 required for specific interaction with the HCV IRES IIIabc domain. X-ray crystal structures of this critical IRES domain bound to eIF3 will result in the first high-resolution structures of the HCV IRES IIIabc domain in a complex with any part of eIF3.